Reductions in Calcium Signaling Limit Inhibition to Diabetic Retinal Rod Bipolar Cells

PURPOSE. The balance of neuronal excitation and inhibition is important for proper retinal signaling. A previous report showed that diabetes selectively reduces light-evoked inhibition to the retinal dim light rod pathway, changing this balance. Here, changes in mechanisms of retinal inhibitory synaptic transmission after 6 weeks of diabetes are investigated. METHODS. Diabetes was induced in C57BL/6J mice by three intraperitoneal injections of streptozotocin (STZ, 75 mg/kg), and confirmed by blood glucose levels more than 200 mg/dL. After 6 weeks, whole-cell voltage-clamp recordings of electrically evoked inhibitory postsynaptic currents from rod bipolar cells and light-evoked excitatory postsynaptic currents from A17-amacrine cells were made in dark-adapted retinal slices. RESULTS. Diabetes shortened the timecourse of directly activated lateral GABAergic inhibitory amacrine cell inputs to rod bipolar cells. The timing of GABA release onto rod bipolar cells depends on a prolonged amacrine cell calcium signal that is reduced by slow calcium buffering. Therefore, the effects of calcium buffering with EGTA-acetoxymethyl ester (AM) on diabetic GABAergic signaling were tested. EGTA-AM reduced GABAergic signaling in diabetic retinas more strongly, suggesting that diabetic amacrine cells have reduced calcium signals. Additionally, the timing of release from reciprocal inhibitory inputs to diabetic rod bipolar cells was reduced, but the activation of the A17-amacrine cells responsible for this inhibition was not changed. CONCLUSIONS. These results suggest that reduced light-evoked inhibitory input to rod bipolar cells is due to reduced and shortened calcium signals in presynaptic GABAergic amacrine cells. A reduction in calcium signaling may be a common mechanism limiting inhibition in the retina.Juvenile Diabetes Research Foundation Postdoctoral Fellowship (New York, NY, USA) [3-PDF-2014-105-A-N]; National Institutes of Health (Bethesda, MD, USA)United States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01-EY026027, 2T32HL7249-36]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Dopamine D1 receptor activation contributes to light-adapted changes in retinal inhibition to rod bipolar cells.

Dopamine modulation of retinal signaling has been shown to be an important part of retinal adaptation to increased background light levels, but the role of dopamine modulation of retinal inhibition is not clear. We previously showed that light adaptation causes a large reduction in inhibition to rod bipolar cells, potentially to match the decrease in excitation after rod saturation. In this study, we determined how dopamine D1 receptors in the inner retina contribute to this modulation. We found that D1 receptor activation significantly decreased the magnitude of inhibitory light responses from rod bipolar cells, whereas D1 receptor blockade during light adaptation partially prevented this decline. To determine what mechanisms were involved in the modulation of inhibitory light responses, we measured the effect of D1 receptor activation on spontaneous currents and currents evoked from electrically stimulating amacrine cell inputs to rod bipolar cells. D1 receptor activation decreased the frequency of spontaneous inhibition with no change in event amplitudes, suggesting a presynaptic change in amacrine cell activity in agreement with previous reports that rod bipolar cells lack D1 receptors. Additionally, we found that D1 receptor activation reduced the amplitude of electrically evoked responses, showing that D1 receptors can modulate amacrine cells directly. Our results suggest that D1 receptor activation can replicate a large portion but not all of the effects of light adaptation, likely by modulating release from amacrine cells onto rod bipolar cells. NEW & NOTEWORTHY We demonstrated a new aspect of dopaminergic signaling that is involved in mediating light adaptation of retinal inhibition. This D1 receptor-dependent mechanism likely acts through receptors located directly on amacrine cells, in addition to its potential role in modulating the strength of serial inhibition between amacrine cells. Our results also suggest that another D2/D4 receptor-dependent or dopamine-independent mechanism must also be involved in light adaptation of inhibition to rod bipolar cells.NIH National Eye Institute [RO1-EY026027]; International Retinal Research Foundation (IRRF) grant; NSF CAREER award [1552184]; University of Arizona NIH Interdisciplinary Training in Cardiovascular Research grant [4T32HL007249-40]12 month embargo; first published May 30, 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Upregulation of synaptotagmin IV inhibits transmitter release in PC12 cells with targeted synaptotagmin I knockdown

Abstract Background The function of synaptotagmins (syt) in Ca2+-dependent transmitter release has been attributed primarily to Ca2+-dependent isoforms such as syt I. Recently, syt IV, an inducible Ca2+-independent isoform has been implicated in transmitter release. We postulated that the effects of syt IV on transmitter release are dependent on the expression of syt I. Results To test this, we increased syt IV expression in PC12 cells by either upregulation with forskolin treatment or overexpression with transfection. Two separately generated stable PC12 cell lines with syt I expression abolished by RNAi targeting were used and compared to control cells. We measured catecholamine release from single vesicles by amperometry and neuropeptide Y release from populations of cells by an immunoassay. In syt I targeted cells with forskolin-induced syt IV upregulation, amperometry measurements showed a reduction in the number of release events and the total amount of transmitter molecules released per cell. In cells with syt IV overexpressed, similar amperometry results were obtained, except that the rate of expansion for full fusion was slowed. Neuropeptide Y (NPY) release from syt I knockdown cells was decreased, and overexpression of syt IV did not rescue this effect. Conclusions These data support an inhibitory effect of syt IV on release of vesicles and their transmitter content. The effect became more pronounced when syt I expression was abolished.</p

Release of Ubiquitin-Charged Cdc34-S‫ف‬Ub from the RING Domain Is Essential for Ubiquitination of the SCF Cdc4 -Bound Substrate Sic1

case of HECT (homology to E6-AP C terminus)-typ

Relative Strengths of Relationships Between Plant, Microbial, and Environmental Parameters in Heavy-Metal Contaminated Floodplain Soil

We used a combination of sampling and statistical approaches to investigate the relative influence of metals, soil acidity, and organic matter on a suite of analogous plant and microbial community parameters in floodplain soils contaminated by mine wastes in the early twentieth century. We compared the sensitivity of plant and microbial communities to environmental variables and to one another using constrained ordination analyses. Environmental factors accounted for a larger percentage of the total variance in microbial communities (56.2%) than plant communities (22.0%). We also investigated biological and geochemical changes that occurred along a short transect (64 cm) that spanned a transition from productive grassland to an area of barren wasteland representing a total functional collapse of the grassland/soil ecosystem. Along this small-scale transect we quantified geochemical parameters and biological parameters in two soil layers, an upper layer (0–10 cm) and a lower layer (10–20 cm). Results from the short transect indicated that soil respiration was not a strong indicator of underlying metal concentrations, but soil acidity was correlated in the upper and lower layers. PLFA profiles changed with distance along the gradient in the upper, but not the lower layer. Implications for remediation of contaminated floodplain soils are discussed